For decades, we have known that loss of acetylcholine (ACh) and cholinergic markers is a hallmark of cognitive decline in aging. Yet, pro-cholinergic medications, the most widely used palliative treatments, have only a modest (at best) effect on cognitive symptoms. Improving therapeutic efficacy requires a detailed accounting of cholinergic changes in aging (Aim 1), deeper insight into the structural underpinnings of these changes and whether functional modifications (e.g. therapeutic action) can compensate for them (Aim 2), and an early marker of decline, signaling the need for intervention (Aim 3). Using a translational approach, with parallel analysis in rodents and humans, this is the information our study will provide. Rodent studies with transgenic mouse models allow for visualization and quantification of cholinergic cell bodies and projection fields in health and models of Alzheimer?s disease (AD). Using these models, we have shown that the long, highly branched cholinergic projections and their terminations are particularly vulnerable, and therefore likely compromised early in cognitive decline. Further, we are one of the few institutions that have regulatory approval for, and have synthesized, the new radiotracer, [18F]VAT. This tracer binds specifically to the vesicular ACh transporter (VAChT; protein that packages ACh into vesicles), a sensitive marker for cholinergic function in vivo. We have performed both clinical (PET) and preclinical (microPET) imaging with this tracer, using pilot funding from the Alzheimer?s Foundation of America. Our compelling pilot data in humans reveals a significant relationship between cognitive ability and VAChT density in the entorhinal cortex (EC), a region known to be affected early in AD. In Aim 1, we will quantify this association in rodents and humans, specifically focusing on spatial recognition memory, which is EC-dependent and compromised early in AD. The human cohort will have a spectrum of cognitive deficits -- controls through moderate cognitive impairment. Spatial recognition memory will be assessed by an Object in Place (OiP) task administered by an expert neuropsychologist. The carefully designed translational analogue of these studies in rodents is a cohort of control and AD rodent models at 3 and 6 months. Rodent cognition will be assessed through a displaced object recognition task, which we have previously validated to assess spatial recognition memory. We hypothesize decreased [18F]VAT uptake (microPET and PET) will be correlated to impaired spatial recognition memory. All human participants will also receive amyloid beta imaging, to relate AD pathophysiology to cholinergic tone. In Aim 2, we use the rodent model to uncover the mechanism by which changes to the cholinergic system observed in Aim 1 can affect cognition and whether they can be functionally reversed (mimicking therapeutics). EC structure will be probed with high throughput, high-resolution microscopy and novel, targeted genetic probes to create 3D maps of EC cholinergic terminal fields. This will be related to function by examining the system response to optogenetic modulation and electrophysiology. By probing cholinergic structure and function changes in aging and cognitive decline, we will shed light on the time course of cholinergic deficits. We examine whether these deficits are predictive of AD conversion in Aim 3. Such studies are critical to develop the next generation medications for cognitive impairment, a growing worldwide need.